Mutations in sigma 70 transcription factor improves expression of functional eukaryotic membrane proteins in Escherichia coli

Abstract

Eukaryotic integral membrane proteins (IMPs) are difficult to study due to low functional expression levels. To investigate factors for efficient biogenesis of eukaryotic IMPs in the prokaryotic model organism Escherichia coli, important, e.g., for isotope-labeling for NMR, we selected for E. coli cells expressing high levels of functional G protein-coupled receptors (GPCRs) by FACS. Utilizing an E. coli strain library with all non-essential genes systematically deleted, we unexpectedly discovered upon whole-genome sequencing that the improved phenotype was not conferred by the deleted genes but by various subtle alterations in the "housekeeping" sigma 70 factor (RpoD). When analyzing effects of the rpoD mutations at the transcriptome level we found that toxic effects incurred on wild-type E. coli during receptor expression were diminished by two independent and synergistic effects: a slower but longer-lasting GPCR biosynthesis and an optimized transcriptional pattern, augmenting growth and expression at low temperature, setting the basis for further bacterial strain engineering.

Figure 1

Expression of NTR1 receptor in the selected E. coli strains from the Keio collection. E. coli strain BW25113 (WT) and the most abundant clones of the selected Keio clones were transformed with the plasmid pRG-NTR. Expressions of NTR1 receptor are as described in Materials and Methods. The x-axis label indicates the gene that is deleted on the respective Keio clone. Means and Standard Deviations from three independent experiments are shown. (a) Growth was estimated with OD_600nm measurements after 20 hours of GPCR expression at 20 °C. Strains with statistically significant increases in growth versus wild-type E. coli BW25113 are calculated by two-tailed paired t test; p values are indicated. (b) Receptor expression levels were assessed by radioligand binding assays. The y-axis represents the number of functional receptors per cell, averaged across an entire population of cells. Statistical p values, as calculated by two-tailed paired t test, are indicated for strains with statistically significant increases in receptors per cell versus wild-type E. coli BW25113.

Figure 2

Structural interactions of sigma 70 factor in the RNA polymerase transcription initiation complex and location of selected mutations. (a) Domain organization, conserved regions and promoter recognition of the sigma 70 factor (green). Interactions between sigma 70 and DNA are depicted with dashed lines. The non-template DNA strand is colored magenta and the template strand cyan, with key consensus promoter elements contacted by sigma 70 (light green) indicated in pink with letters. Transcription initiates at +1. Mutations found in the rpoD gene in this work are highlighted in red. Adapted from. (b) Overall structure and organization of E. coli sigma 70 in the RNA polymerase transcription initiation complex based on PDB ID: 4YLN. Sigma 70 (surface representation) and promoter DNA (cartoon) are colored as above. Inset zooms show the HTH domain of sigma 70 where the mutation E575V is located, and interaction of mutations in sigma 70 domain 2 with DNA. Adapted from.

Figure 3

Expression of NTR1 in different strains. E. coli BW25113 (WT), Keio clones and the newly created strains were transformed with the plasmid pRG-NTR. (a) Growth was estimated with OD_600nm measurements after 20 hours of NTR1 expression at 20 °C. (b) Receptor expression levels were assessed by radioligand binding assays. Means and standard deviations from three independent experiments are shown.

Figure 4

Cell growth after 20 h of GPCRs expression in different Keio clones. E. coli BW25113 (WT) and selected Keio clones were transformed with pRG plasmid derivatives encoding the wild-type version of ADRA1b, TACR1 and MOR GPCRs. Growth was estimated with OD_600nm measurement after 20 hours of GPCR expression at 20 °C. The x-axis label indicates the gene that is deleted in the respective Keio clone. Means and standard deviations from three independent experiments are shown. Statistical p values, as calculated by two-tailed paired t test, are indicated for strains with statistically significant increases in growth versus wild-type E. coli BW25113.

Figure 5

Kinetics of NTR1 expression. E. coli BW25113 (wt) and the rpoD E757V mutant strain were transformed with the plasmid pRG-NTR. NTR1 expression at 20 °C was performed for 20 hours. Samples were taken at different time points. (a) Growth was monitored with OD_600nm measurements. (b) Functional receptor levels were estimated by radioligand binding assays. (c) mRNA NTR1 levels were calculated by quantitative real-time PCR and expressed as log₂ ratios between rpoD mutant vs. wt E. coli strain. (d) NTR1 protein levels were monitored with western blots using an anti-MBP antibody (in duplicates). The blot image has been cropped for conciseness. The band intensity data are not quantitative. The full-size blot is presented in Fig. S13.